Cloning, Purification and Characterization of Acetyl Xylane Esterase from Anoxybacillus flavithermus DSM 2641T with Activity on Low Molecular-Weight Acetates

Abstract

Family 4 carbohydrate esterases (CE-4) have deacetylate different forms of acetylated poly/oligosaccharides in nature. This family is recognized with a specific polysaccharide deacetylase domain assigned as NodB homology domain in their secondary structure. Most family 4 carbohydrate esterases have been structurally and biochemically characterized. However, this is the first study about the enzymological function of pdaB-like CE4s from thermophilic bacterium Anoxybacillus flavithermus DSM 2641(T). A. flavithermus WK1 genome harbors five putative CE4 family genes. One of them is 762 bp long and encodes a protein of 253 amino acids in length and it was used as reference sequence in this study. It was described as acetyl xylane esterase (AXE) in genome project and this AfAXE gene was amplified without signal sequence and cloned. The recombinant protein was expressed in E. coli BL21 (DE3), purified by nickel affinity chromatography and its purity was visualized on SDS-PAGE. The activity of the recombinant enzyme was shown by zymogram analysis with α-naphtyl acetate as a substrate. The enzyme was characterized spectrophotometrically using chromogenic p-nitrophenyl acetate. Optimum temperature and pH were determined as 50 °C and 7.5, respectively. Km and Vmax were determined as 0.43 mM and 3333.33 U/mg, respectively under optimum conditions. To our knowledge this is the first enzymological characterization of a pdaB-like family 4 carbohydrate esterase from the members of Anoxybacillus genus.

Cloning, expression and biochemical characterization of aβ-carbonic anhydrase from the soil bacteriumEnterobactersp. B13

“…To determine the esterase activity, zymogram staining was performed with minor modifications as described previously 14 at two different temperatures (+4 C and also at RT) on SDS-PAGE (15%). In brief, followed by electrophoresis the gels were incubated in 100 mM Tris-HCl (pH 7.5) including 0.5% Triton X-100, for 6 h at +4 C and for 4 h at RT.…”

In silico screening and experimental analysis of family GH11 xylanases for applications under conditions of alkaline pH and high temperature

“…Malectin [39] and Ricin B lectin domain [40] have carbohydrate recognition function. GH11 is also found associated with other catalytic domains active against different constituents of the plant cell wall, such as the polysaccharide deacetylase domain [41], the esterase domain [42] and the lipase GDSL domain [43].…”

Comparison of Single-Step Methods to Enrich Lipase Concentrations in Bacterial Cell Lysates

“…Ekinci et al [12] reported an 85% yield and 1.3-fold purification of a lipase from G. stearothermophilus AH22 using 70°C. Also, heat treatment was used to purify a lipase from Staphylococcus aureus, and the yield was 74.6%, and the purification fold was 1.57-fold using 55°C [32].…”